The long-term objective of our research centers on elucidation of the mechanisms whereby critical cell cycle 'regulators contribute to the genesis and progression of neoplastic transformation. Much of our current work focuses on how the contribution of regulated, ubiquitin-dependent destruction of cyclin D1 for normal versus neoplastic growth. The noted overexpression of cyclin D1 in more than 50% of human esophageal cancer highlights the importance of elucidating the mechanisms that regulate cyclin D1 activity in this deadly cancer. While cyclin D1 overexpression is a consequence of gene amplification and chromosome translocation in a subset of cancers, decreased cyclin D1 protein degradation, which depends on its phosphorylation of Thr286, is thought to be the key factor in a majority of esophageal cancers. We have recently identified the SCF(Fbx4-aB crystallin) as the E3 ubiquitin ligase that controls cyclin D1 ubiquitination and degradation. This critical discovery has provided essential information and tools necessary to assess the mechanisms that contribute to regulation of cyclin D1 accumulation during malignant progression. More importantly, our recent discovery of inactivating mutations in Fbx4 in esophageal cancer strongly supports our hypothesis that Fbx4 functions as a novel tumor suppressor gene. The identification of this E3 ligase as well as our recent preliminary studies lead to the overarching hypothesis that the SCF(Fbx4 aB crystallin) ligase via coordinated recognition of phospho-cyclin D1 by Fbx4 and aB crystallin, plays a critical role in the maintenance of esophageal cell proliferation, and trasformation. The hypothesis will be pursued by the (following interrelated Specific Aims: 1) To define the post-translational regulation of cyclin D1 by Fbx4 as it pertains to ligase function, utilizing esophageal cancer derived mutations as a guide; 2) To determine the contribution of Fbx4 to esophageal tissue homeostasis and transformation in vivo and in vitro; and 3) To evaluate the role of Fbx4 in maintenance of genomic stability and response of tumor cells to cheomtherapeutic intervention. Project 3 is closely integrated with Project 1 (Aims 1 and 2) and Project 2 (Aim 2), and utilizes all the outstanding cores. RELEVANCE (See instructions): This Project has made fundamental new discoveries in the regulation of cyclin D1, often overexpressed in esophageal squamous cell cancer. To that end, Fbx4, part of the E3 ligase that regulates cyclin D1, is mutated in this cancer. This has applications in molecular-clinical correlations for cancer prognosis, and developing new strategies for controlling cell cycle regulation and DMA damage responses.